Individuals with locomotive syndrome (LS) require nursing care services owing to problems with locomotion and the musculoskeletal system. Individuals with LS generally have a reduced walking speed compared with those without LS. However, differences in lower-limb kinematics and gait between individuals with and without LS are not fully understood. This study aimed to clarify the characteristics of the gait kinematics of individuals with LS using wearable sensors.We assessed 125 participants (mean age 73.0 ± 6.7 years) who used a public health promotion facility. Based on the 25-question Geriatric Locomotive Function Scale (GLFS-25), these participants were grouped into the non-LS (GLFS-25 < 7), LS-stage 1 (GLFS-25 7–16), and LS-stage 2 (GLFS-25 ≥ 16) groups (larger GLFS-25 scores indicate worse locomotive ability). Spatiotemporal parameters and lower-limb kinematics during the 10-m walk test were analyzed by the “H-Gait system”, which is a motion analysis system that was developed by the authors and is based on seven inertial sensors. The peak joint angles during the stance and swing phases, as well as the gait speed, cadence, and step length were compared among all groups.P = 0.002), hip flexion , and knee flexion . The LS-stage 1 and LS-stage 2 groups had a significantly slower mean gait speed than the non-LS group .There were 69 participants in the non-LS group, 33 in the LS-stage 1 group, and 23 in the LS-stage 2 group. Compared with the non-LS group, the LS-stage 2 group showed significantly smaller peak angles of hip extension (9.5 ± 5.3° vs 4.2 ± 8.2°, The LS-stage 2 group showed significantly different lower-limb kinematics compared with the non-LS group, including smaller peak angles of hip extension, hip flexion, and knee flexion. It would be useful to assess and improve these small peak joint angles during gait for individuals classified as LS-stage 2. With the aging of society, the number of musculoskeletal-related disease cases is rapidly increasing in Japan . Among tLS is diagnosed using the 25-question Geriatric Locomotive Function Scale (GLFS-25) . The GLFGait analysis is used in many clinical settings to diagnose disability and evaluate walking ability. Reduced walking ability in people with LS is reflected by spatiotemporal gait parameters, including gait speed and stride length , 11. ComWe have recently developed a wearable sensor-based three-dimensional motion analysis system called the H-Gait system . The H-GThe participants were recruited from local residents who used a public health promotion facility in Iwamizawa city, Japan. A total of 125 individuals participated in the present study . Participants were included if they were: (1) aged 65 years or above; (2) able to ambulate 10 m independently without walking aids; (3) able to complete the GLFS-25. The exclusion criteria were any acute or uncontrolled cardiac, pulmonary, or musculoskeletal conditions, severe visual impairment, or cognitive impairment. All participants provided written informed consent before participating in the study. This study was approved by the review board of our institution .Participants were divided into the following three groups based on the results of the GLFS-25 and 10-m walk test  with seven wearable sensor units . The first 2 m and the last 2 m of the 10-m walk test were reserved for acceleration and deceleration, respectively. Participants performed two practice trials, followed by two trials of the 10-m walk test at a self-selected speed.The gait assessment protocol was as follows. First, ten spherical markers were attached to the greater trochanters, medial and lateral femoral epicondyles, and medial and lateral malleoli Fig. . To scalThe spherical markers were then removed, and seven sensor units were secured using velcro bands with a pocket at the sacrum, anterior aspect of the mid-thigh, anterior aspect of the mid-shank, and most anterior point of the shoes. Sensor units were synchronized and recorded the tri-axial acceleration and tri-axial gyro sensors with a sampling rate of 100 Hz. Before the 10-m walk test, sensor calibration was performed for each participant in the upright and inclined positions to calculate the initial inclination of each sensor . Before Data analysis was performed using MATLAB software  with a customized motion analysis program. The thigh length, shank length, foot height, and hip width were measured using the three static images . The senEach gait cycle was defined as the motion from one heel contact to the next heel contact of the same foot. Heel contact and toe-off were determined by the peak angular velocity of the shank , 18. PeaP = 0.05. The effect sizes were calculated to determine the magnitude of the differences using eta squared (η2). A sensitivity power analysis revealed a moderate effect size with α = 0.05, power = 0.8, total sample size = 125, and f = 0.28. All statistical analyses were performed using SPSS Statistics 22 .One-way analysis of variance (ANOVA) was conducted to compare the demographics, spatiotemporal gait parameters, and gait kinematics during the 10-m walk test among the groups. Differences in sex ratios among the groups were tested with the chi-squared test. The Tukey HSD test was used for post-hoc pairwise comparisons. The level of significance was set as P < 0.001 and P = 0.006, respectively, η2 = 0.15). The LS-stage 2 group also had a significantly slower cadence  and shorter step length  than the non-LS group. In contrast, the spatiotemporal gait parameters did not differ between the non-LS and LS-stage 1 groups. The trajectory angle of the ankle joint in the horizontal plane was significantly larger in the LS-stage 2 group than the non-LS group ; however, the trajectory angle of the knee joint in the horizontal plane did not differ among the groups.Based on the GLFS-25 results, there were 69 participants in the non-LS group, 33 in the LS-stage 1 group, and 23 in the LS-stage 2 group  and the abduction angle  differed among the groups. Post-hoc testing showed that the peak hip extension angle was significantly smaller in the LS-stage 2 group than in the non-LS group and the LS-stage 1 group (P = 0.002). The peak hip abduction angle was significantly larger in the non-LS group than in the LS-stage 1 group and the LS-stage 2 group . There were no other differences between the groups in the peak joint angles during the stance phase.Kinematic analysis revealed significant differences among the groups Table . During P = 0.018, η2 = 0.06) and the knee flexion angle (P = 0.006 η2 = 0.08) differed among the groups. The peak hip flexion angle was significantly smaller in the LS-stage 2 group than in the non-LS group and the LS-stage 1 group . The peak knee flexion angle was also significantly smaller in the LS-stage 2 group than in the non-LS group and the LS-stage 1 group . There were no differences between the non-LS group and the LS-stage 1 group in the peak joint angles during the swing phase.During the swing phase, the peak hip flexion angle . During the swing phase, individuals with LS-stage 2 had significantly smaller peak flexion angles of the hip and knee joints and a larger peak hip abduction angle than those without LS; the decreased peak hip extension and flexion angles and peak knee flexion angles are consistent with the findings of a previous study that assessed the gait kinematics in people with knee OA , 20. In The LS-stage 2 group had a significantly slower walking speed, and shorter step length than the non-LS group. These results are consistent with those of a previous study that assessed the spatiotemporal gait parameters in patients with knee OA , 21. TheThe LS-stage 2 and LS-stage 1 groups had significantly smaller peak hip abduction angles during the swing phase compared with the non-LS group. However, these differences between groups were within the measurement error range . TherefoIn the present study, the angle between the trajectory lines of the bilateral ankles was significantly greater in the LS-stage 2 group than in the non-LS group. The trajectory line of the ankle in the horizontal plane reflects the combination of the kinematics of abduction–adduction and rotation in the hip and knee joints during each gait cycle . AlthougThe present study has several limitations. First, the classification of the severity of LS was based on the results of the GLFS-25. Previous studies have assessed the severity classification of LS using the Stand-up Test and the 2-Step Test [In the present study, the H-Gait system made it possible to obtain data from a large number of local residents. We were also able to identify the spatiotemporal gait parameters and gait kinematics of LS. This may provide a basis for proposing new intervention methods for the prevention of LS. The changes in gait kinematics with the progression of LS warrant investigation in a prospective cohort study.The present study was the first to investigate the gait kinematics and spatiotemporal gait parameters during walking in subjects with LS using a wearable sensor system. This study showed that subjects with LS-stage 2 had significantly smaller peak hip extension angles during the stance phase compared with subjects without LS. During the swing phase, subjects with LS-stage 2 had significantly smaller peak flexion angles of the hip and knee joints and a larger peak hip abduction angle compared with subjects without LS. It would be useful to improve the small peak joint angles during gait for individuals with LS-stage 2. Individuals with LS-stage 2 who perform increasing hip extension angle and hip flexor strength training may show improvements in their walking ability.


Significant increase in miR-182-5p and miR-183-5p expressions, as well as a significant decrease in FOXO1 and miR-30d-5p expressions, suggest that PTC cells may tend to preserve their autophagy capability. Papillary thyroid carcinoma (PTC) is the most common endocrine cancer. However, the role of biomechanics in the development and progression of PTC is obscure. The microarray dataset GSE104005 was examined to identify important microRNAs (miRNAs or miRs) and their probable roles in the carcinogenesis of PTC. The gene expression omnibus (GEO) database was used to obtain the data. R was used to access the differentially expressed miRNAs (DEMs) and genes (DEGs). The multiMiR software was used to predict DEM targets. To validate the top DEMs and DEGs, thirty tissue samples were obtained from PTC patients who had their thyroids removed and compared with 30 normal samples. The total RNA content of the tumor and corresponding non-tumoral adjacent samples were purified and converted to cDNA. Expression levels of top dysregulated miRNAs and their target and predicted DEG were evaluated using the RT-qPCR method. miR-182 and miR-183 were top upregulated miRs and miR-30d was the most downregulated miR among DEMs. Furthermore, FOXO1 which was shown to be targeted by aforementioned miRNAs, was the most downregulated genes among other DEGs. 10 hub nodes were detected by PPI construction. PTEN was the hub node with highest score. The i Thyroid gland is an endocrine organ with a remarkable risk of cancer. Thyroid cancer has become more common in recent decades . Almost Data collection and preprocessing: The microRNA expression profile GSE104005 was downloaded from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih. gov/geo/). Quantile normalization was performed to normalize the dataset. This dataset, based on Illumina HumanHT-12 WG-DASL V4.0 R2 expression beadchip, was made up 29 thyroid carcinoma samples and 5 non-neoplastic thyroids. In this case, we included 20 PTC samples as our inclusion criteria and compare the miRNA expression profiles with control samples. Identification of DE-miRNAs and DEGs: R software, version 4.1.1, was used to analyze the data. Quantile normalization approach was used to normalize the data in this case. The Gplot software is also used to create boxplots using normalized data. To detect DE-miRNAs and DEGs, the Limma (version 3.483), data.table (version 1.14), and GEOquery (version 2.60) packages were downloaded and utilized. In our analysis, p< 0.05 and |Log2FC| > 1 were used as cutoff criterion. Finally, the Pheatmap (version 1.0.12) and Enhancedvolcano (version 1.0.10) packages were used to display the heatmap and volcano diagram, respectively. Identification of target genes: Once we have a list of possible miRNAs, the next step is to determine the target genes of these miRNAs so that we may run further analyses based on them, such as gene ontology. Multimir package which predicts target genes from mirTarbase and Tarbase databases, was used to anticipate target genes and in common genes between two databases were elucidated and illustrated by Venndiagram package. The procedures taken result in the identification of a large number of genes that may be utilized to discover molecular and cellular mechanisms in the progression of PTC. Functional Enrichment Analysis of Putative Target Genes: To supplement Gene Ontology (GO) and KEGG pathway analyses, identified target genes were uploaded to Funrich 3.1.3 software p 0.05 was used as the inclusion criterion for both GO and KEGG. GO is a popular method for linking genes, gene products, and sequences to biological phenomena. KEGG is a database that allows scientists to analyze genomic sequences and other high-throughput biological data.PPI network construction: Following functional enrichment analysis, a protein-protein interaction network was constructed using STRING and Cytoscape software. The target gene was selected from the STRING database with a score of >0.9, and the interaction network was visualized using Cytoscape software. The CytoHubba app was also used to identify hub genes. The hub genes are a small number of genes with a large number of interaction partners that serve as an important node in the PPI network for gene interaction. By overlaying the top 10 genes, CytoHubba discovered hub genes using 11 categorization algorithms.Sample collection: The demographic information of patients is summarized in RNA Extraction, cDNA synthesis, and reverse transcription-quantitative (RT-q) PCR: Total RNA was extracted from the 300 mg purified human PTC samples using Trizol reagent  according to the manufacturer's protocol. Nanodrop device (Spectrophotometer 2000/2000c) was used to assess the quantity of purified RNA (ng/ul) and any protein or mineral residual contaminations. Complementary DNA synthesis was performed using AnaCell cDNA synthesis kit (Lot No:cs0025) following the manufacturer's temperature protocol: 70°C for 5 min, 37°C for 60min, and 70°C for 5 min. Biomir high sensitivity microRNA kit (Zistroyesh Co/Iran) provided stem-loop technology for complementary DNA synthesis for each target and housekeeping miRNA (U6). PCR, using miR-specific primers and FOXO1 primer was performed using SYBER Green PCR kit (AnaCell/Iran). Stem-loop RT amplification primers for miRNAs were provided by the kit (Zistroyesh Co/Iran) according to the manufacturer's instruction. The PCR conditions were as follows: 95°C pre-denaturation for 15 min, followed by 40 cycles of denaturation at 95°C for 20 seconds, an annealing-extension step at 60°C for 60 seconds, followed by a melt analysis step between 55°C and 95°C. The housekeeping gene to normalize the FOXO1 and miRNAs was the GAPDH  and U6 gene, respectively. All RT-qPCR assays were complemented triplicate. Primer details for FOXO1 and GAPDH housekeeping gene are tabled in Statistical Analysis: Gene expression analysis was performed using ΔΔCt method, where ΔΔCt = ΔCt  -  [control) . To compAs shown in On the aforementioned possible target genes, GO functional and KEGG pathway enrichment analysis were conducted. The following tables shows the enriched GO functions for the target genes, which include transcription regulatory activity in the (MF) category , nucleosIn addition to determining the activities of protein products derived from the analyzed genes, the interaction of these products was investigated, revealing a huge network of molecular linkages between proteins. As shown in the By analysis of FOXO1 mRNA levels in the PTC and adjacent normal tissues, a 2.66-fold decrease in the expression levels of FOXO1 was observed in the PTC samples in comparison with adjacent normal tissues. This outcome was statistically significant (p<0.0001). When miR-182-5p levels were compared to non-cancerous neighboring samples, there was a substantial significant 1.73-fold increase in the expression level of this miRNA in the PTC (p=0.007). The examination of miR-183-5p expression levels in PTC and normal surrounding tissues, revealed that this miRNA was expressed significantly 2.35-fold higher in PTC samples (p=0.0001).in By comparing the expression levels of miR-30d-5p in PTC and neighboring normal tissues, we discovered a 2.62-fold significant decrease in the expression of this miRNA in PTC samples compared to adjacent normal tissues  pathway were significantly enriched in target genes of dysregulated DEMs, according to Go and KEGG analysis results. The involvement of hsa-mir-182 , 15, hsaIn an address to transcription factor-independent activity of FOXO1, our findings indicate a 2.66-fold (p<0.0001) significant decrease in mRNA level of FOXO1 in PTC compared to normal adjacent (control) tissues suggesting an overall insufficiency in the related tumor suppressor pathways. This reduction can be explained by our results, including the miR-182-5p and miR-183-5p significant increase in expressions of 1.73- (p=0.007) and 2.35-fold (p<0.0001), respectively. The correlations of these alterations in tumor tissues are not statistically significant and this finding can be attributed to the small sample sizes (30 for both PTC and non-cancerous tissues). However, using the DIANA-TarBase v8 experimentally supported miRNA targets database, the prediction score of these two miRNAs suppressing FOXO1 is estimated as 0.821 and 0.783 out of 1 for miR-182-5p and miR-183-5p, respectively. Therefore, upregulation of these miRNAs, may indicate the epigenetic regulation of the FOXO pathway. According to this inhibitory effect of these two miRNAs on the FOXO1 tumor suppressor gene and by considering their positive effect on tumor progression, this data can be used to design future studies to bring up the repression of miR-182-5p and miR-183-5p as a part of PTC treatment methods , 27. Interestingly, previous studies demonstrate that the ATG5 gene plays an important role in establishing autophagy . This geDespite our hypothesis about the beneficial impact of autophagy in PTC, recent investigations indicate an inverse relationship between autophagy and PTC progression. For instance, it is demonstrated that miR-524-5p enhances autophagy by targeting FOXO1 mRNA resulting in the PTC development suppression . It is aCollectively, our findings of a significant decrease in the expression levels of FOXO1 and miR-30d-5p in the PTC compared to normal adjacent tissues suggest that the PTC cells may tend to preserve their autophagy activity by considering the role of autophagy in the cancer cell homeostasis. These data were gained by bioinformatic tools and validated in vitro. Therefore, this concept in parallel to other studies of autophagy may be utilized as potential items for future PTC research. Furthermore, our data can be used by future investigations.This research is financially supported by Tabriz Hematology and Oncology Research Center.The authors have stated that they have no possible conflicts of interest in respect to this publication to disclose.


Traditional temperature detection has limitations in terms of sensing accuracy and response time, while chip-level photoelectric sensors based on the thermo-optic effect can improve measurement sensitivity and reduce costs. This paper presents on-chip temperature sensors based on polysilicon (p-Si) waveguides. Dual-microring resonator (MRR) and asymmetric Mach–Zehnder interferometer (AMZI) sensors are demonstrated. The experimental results show that the sensitivities of the sensors based on AMZI and MRR are 86.6 pm/K and 85.7 pm/K, respectively. The temperature sensors proposed in this paper are compatible with the complementary metal-oxide-semiconductor (CMOS) fabrication technique. Benefitting from high sensitivity and a compact footprint, these sensors show great potential in the field of photonic-electronic applications. Silicon photonics are a promising solution for large-scale and high-performance photonic integrated circuits (PICs) with a growing amount of applications in the optical interconnections ,2, progrNowadays, most silicon photonic devices and circuits are demonstrated on the silicon-on-insulator (SOI) platform ,15,16. C2 buffer at 2 μm of thickness at 620 °C. The sensor is designed for C-band operation. For single-mode propagation, the geometry of the waveguide is 500 nm × 220 nm. Deep ultraviolet (DUV) photolithography was employed to define the waveguide patterns, followed by inductively coupled plasma (ICP) etching of silicon. To simplify the fabrication process, we chose one-step etching of 150 nm for both grating couplers and ridge waveguides. A 1 μm thick SiO2 upper cladding was deposited on the waveguides. The devices were fabricated with the 180 nm p-Si photonic multi-project-wafer (MPW) process at the Institute of Microelectronics of the Chinese Academy of Sciences (IMECAS). The 220 nm thick p-Si was deposited on the SiOMRRs are usually compact with a typical radius of several micrometers. The narrow linewidth of resonance dip is attractive for highly sensitive optical sensors . Howeverload = λ/Δλ of 3370.Our dual-MRR sensor consists of a ring (MRR1) and a racetrack (MRR2) resonator. The gap between the ring and bus waveguide is 300 nm. The racetrack resonator consists of a directional coupling (DC) coupling region. For the coupling region, the gap and the length are 400 nm and 10 μm, respectively. The radii of the ring and racetrack are 30 μm and 40 μm, respectively. A broadband tunable laser system (Santec Full-band TSL-550) covering the ultra-wide tuning range of 1260 nm to 1630 nm was used to characterize the fabricated devices. The resolution is 1 pm for MRR, narrow linewidth components, in the measurements. This system combines up to three tunable lasers (TSL-550) with an optical switch module (OSA-110). The light from output grating is measured through an optical power meter (MPM-210H). The light from laser and devices is coupled into the devices through a vertical fiber coupling system with a polarization controller. The minimum loss of the reference grating is ~10.42 dB for the fundamental transverse electric (TE) mode. The spectrum of the dual-MRR sensor from 1543 nm to 1568 nm at 25 °C is shown in 2) of 0.99996.The temperature response of the sensor was investigated by placing it on a high-precision temperature console stage with a temperature resolution of 0.1 K. Mach–Zehnder Interferometers (MZIs), one of the most widely used structures, have been demonstrated in a wide range of applications, including wavelength division multiplexers ,35, optiThe 1 × 2 MMI used in the sensor is shown in g is the group index, and ∆L is the path length difference between the arms. The group index ng is 4.11, which is calculated through finite difference eigenmode (FDE) method. The center wavelength λ is 1550 nm. The interferometer path length difference ∆L is 280 μm. Thus, the FSR of AMZI is 2.08 nm.The FSR is also important for an AMZI, which is given byThe same characterization system with a dual-MRR sensor is applied for the AMZI. The transmission spectrum of our designed AMZI sensor is shown in 2) of 0.998. Therefore, the AMZI sensor has a sensitivity of 86.66 pm/K.We chose the resonant wavelength dip at 1549.07 nm to demonstrate the sensing application. The drift of the characteristic wavelength observes the temperature change. The performance comparison between temperature sensors on different platforms is listed in load of dual-MRR is 3370. We optimized the 1 × 2 MMI of the AMZI sensor with an excess loss of 1.54 dB and uniform spectral uniformity. For both sensors, we used the shift of the resonant wavelengths to calculate the amount of temperature change. The results show the sensitivity of the dual-MRR sensor and AMZI sensor are 85.74 pm/K and 86.6 pm/K, respectively. It confirms that the temperature sensors based on the p-Si waveguides have large temperature sensitivity and a compact footprint. Therefore, they show great potential to realize temperature monitoring of multilayer integrated optical circuits and EPICs.In conclusion, we demonstrated two types of p-Si temperature sensors experimentally. The two types of sensors were dual-MRR and AMZI structures. The dual-MRR sensor had a large FSR of 14.56 nm and the Q


P < .001). The overall mean CSA at the last follow-up increased over time in both the groups compared with the post-operative CSA (P = .003). The fusion rate at 1 year after surgery was 86.8% and 82.9% in the subsidence and non-subsidence groups, respectively. However, the differences in the SA, CSA, and fusion rates between the groups were not statistically significant . Subsidence after stand-alone ACDF occurs to a certain capacity; however, it does not appear to significantly influence the radiological and clinical outcomes if foramen decompression is adequately and sufficiently provided in a long-term follow-up study. In contrast, subsidence appears to positively affect the fusion rate in the short-term follow-up.This study aimed to evaluate the influence of subsidence in patients who performed stand-alone anterior cervical discectomy and fusion (ACDF) by analyzing the long-term clinical and radiological outcomes. This retrospective study enrolled 53 patients with 79 segments with degenerative cervical disease treated with stand-alone ACDF with ≥5 years of follow-up. Segmental angle (SA), cervical sagittal alignment (CSA), subsidence, and fusion were analyzed. Visual analog scale (VAS) scores and neck disability index (NDI) were also evaluated. Subsidence occurred in 24 (45.2%) patients and 38 segments (48.1%) at the last follow-up. The mean VAS score and NDI had improved in both the subsidence and non- subsidence groups. The mean SA at the last follow-up had increased to 1.3° ± 8.5° in the subsidence group and to 1.5° ± 5.2° in the non-subsidence group compared with the post-operative SA ( Meanwhile, the most critical issue of stand-alone ACDF is subsidence, which can cause cervical kyphotic changes or clinical deterioration. According to some studies, cage subsidence occurred in 19% to 63% of the patients after stand-alone ACDF.–12 Moreover, some studies have demonstrated that stand-alone ACDF was unsatisfactory, with worse radiological outcomes noted due to subsidence, compared with standard ACDF with plate fixation.,14 In contrast, some studies have reported that cage subsidence was not significantly associated with clinical and radiological outcomes.,16Anterior cervical discectomy and fusion (ACDF) with plate fixation has been recommended for degenerative cervical disease involving two or more levels, with instability and kyphosis.This study aimed to evaluate the influence of subsidence in patients who underwent stand-alone ACDF using polyether ether ketone (PEEK) cages without plate fixation by analyzing the long-term clinical and radiological outcomes.This study was performed according to the requirements associated with patient anonymity and was approved by the Institutional Review Board (CNUH-2020-054).We conducted a retrospective review of 202 patients with degenerative cervical disease who performed ACDF with or without plate fixation performed by a single neurosurgeon between 2009 and October 2015 at our institution. The inclusion criteria were as follows: patients with cervical radiculopathy or cervical spondylotic myelopathy unresponsive to medical treatment; patients treated with one-, two-, or multiple-level stand-alone ACDF; patients with a clinical and radiological follow-up of ≥60 months after surgery. The exclusion criteria were as follows: history of cervical spine surgery; presence of other cervical diseases, including infections or tumors; patients with clinical or radiological follow-up of <60 months after surgery.In total, we enrolled 53 study patients with degenerative cervical disease who performed stand-alone ACDF with a follow-up duration of ≥60 months. To identify the patients’ clinical characteristics, the clinical data at the time of treatment  were collected.R; Hansbiomed Corp, Seoul, South Korea), intermixed with autologous bone chips, and inserted into the disc space. The operation was completed without any anterior plating. All the patients wore a Philadelphia cervical collar  for 8 weeks.General anesthesia was used for all the patients. The standard Smith–Robinson method was used to expose the involved segment. After exposing the affected prevertebral space, the alignment and affected levels were confirmed by intraoperative fluoroscopy. After discectomy, the posterior annulus, posterior longitudinal ligament, and osteophytes were removed. In most cases, uncoforaminotomy was sufficiently performed to totally decompress the nerve roots. The upper and lower cartilaginous endplates were decorticated, preserving the bony endplates. Local autologous bone chips were collected during removal of osteophytes for grafting. The appropriate PEEK cage was selected , and the cages were filled with a demineralized bone matrix , cervical sagittal alignment (CSA), and subsidence were measured using lateral plain radiographs. The SA was measured using the Cobb angle, that is, the angle between the superior endplate of the upper adjacent vertebra and the inferior endplate of the lower adjacent vertebra at the operated disc level. The overall CSA was measured according to the Cobb angle between the lower endplate of C2 and lower endplate of C7. Kyphosis was noted as a positive value, whereas lordosis was noted as a negative value. The total intervertebral height (TIH) was measured at the anterior, middle, and posterior points of the superior endplate of the upper adjacent vertebra and the inferior endplate of the lower adjacent vertebra at the operated disc level   Fig. . Based oTo evaluate pain in the neck and arms, a visual analog scale (VAS) was used pre- and post-operatively. The neck disability index (NDI) questionnaire was administered pre- and post-operatively and at the last follow-up.t test was used to evaluate the results pre- and post-operatively. The independent t test and Mann–Whitney U test for parametric variables were used for analyzing relationships between the clinical and radiological outcomes and subsidence. Quantitative data are presented as mean and standard deviation and qualitative data as frequency or percentage. A P value of <.05 was considered to be statistically significant.We analyzed the radiological and clinical outcomes between the subsidence and non-subsidence groups. Data were entered into SPSS version 25.0 . The chi-square test was used to compare qualitative data between the groups. One way analysis of variance was used for quantitative data. A paired samples This study examined 79 treated segments in 53 patients. The mean age of the patients was 60  years at the time of the stand-alone ACDF. There were 36 (67.9%) male and 17 (32.1%) female patients. Further, 29 (54.7%), 22 (41.6%), and 2 (3.7%) patients underwent stand-alone ACDF surgery at one, two, and three segments, respectively. Stand-alone ACDF was performed more at the C4–5 level , followed by the C5–6 , C6–7 , and C3–4  levels. The mean follow-up period after surgery was 89  months. The baseline and pre-operative clinical characteristics of the enrolled patients are presented in Table P < .001) (Table P < .001). The mean CSA at the last follow-up increased significantly in both the groups compared with the values recorded post-operatively (P = .003). However, the mean SA and CSA difference between the groups were not statistically significant . The fusion rate at 1 year after surgery was 86.8% in the subsidence group (33 of 38 segments) and 82.9% in the non-subsidence group (34 of 41 segments) on segmental analysis, and the difference in the fusion rates between the two groups was not statistically significant (P = .682) (Table Fusion was achieved for 67 segments (84.8%) at 1 year post-operatively and for 79 segments (100%) at the last follow-up. Additional surgical treatments for fusion were not required during the follow-up period. Subsidence occurred in 38 segments (48.1%) in 24 (45.2%) patients at the last follow-up. We divided the patients into the subsidence and non-subsidence groups for a comparative analysis. There were 18 men and six women with a mean age of 54.3  years in the subsidence group and 18 men and 11 women with a mean age of 50.9  years in the non-subsidence group. The mean follow-up period was 75.5  months and 83.8  months in the subsidence and non-subsidence groups, respectively. No significant difference was noted in the number of segments involved (single- vs multi-level) or the cage material used (Solis cage vs. Cornerstone cage) between the two groups (Table P < .001) at the last follow-up, and no significant difference was noted between the groups (P = .441). The overall pre-operative VAS score for arm pain was 6.8 ± 1.3 and 3.5 ± 1.8 at the last follow-up. Similarly, the VAS score had improved significantly to 3.2 ± 1.1 and 3.5 ± 1.3 in the subsidence and non-subsidence groups, respectively (P < .001) at the last follow-up, with no significant difference between the groups (P = .631). Furthermore, the NDI had improved over time during the follow-up period in both the groups (P < .001), but the difference was not statistically significant (P = .705) ; they suggested that the subsidence progressed over time continued until the last follow-up. In addition, the VAS scores for neck and arm increased. When patient satisfaction was assessed at the last follow-up using Odom’s criteria, 64.6% of the patients selected “unsatisfactory” response. However, a significant association was not found between subsidence and the clinical outcomes; there was no long-term follow-up in all the patients, and the surgeries were not performed by a single surgeon in their study. Several previous reports have demonstrated that the subsidence did not significantly influence the clinical outcomes, and that subsidence mostly occurred within 1 year after surgery and stabilized subsequently.–16,25 In our study, although the prevalence of subsidence was 48.1%, neck and arm pain had reduced, and the NDI had improved at the last follow-up (P < .001). Although radiating arm pain in one patient in the subsidence group recurred, it improved after posterior cervical foraminotomy, and the incidence rate was not high . It may be assumed that the foraminal height also decreases with subsidence, thereby affecting the clinical outcome. Therefore, we suggest that subsidence does not influence the clinical outcome if foramen decompression is performed adequately and sufficiently during surgery. In addition, our follow-up of 5 years supported the findings of previous reports that a decrease in the intervertebral height mostly occurred within 1 year after surgery; thereafter, the tendency of decrease in the intervertebral height was negligible until the last follow-up . Regarding cervical stability, although the changes in the SA and CSA from the post-operative period to the last follow-up were larger in the subsidence group than in the non-subsidence group, the difference between the groups was not statistically significant. Thus, it can be assumed that the pre-operative SA and CSA may affect the changes in the post-operative SA and CSA. Consequently, stand-alone ACDF without anterior plate fixation does not appear to be the only factor involved in the deterioration of the SA and CSA.To date, several studies have reported that stand-alone ACDF acquired a successful fusion rate of >90%.,16 Subsidence is an unavoidable consequence of stand-alone ACDF; if foramen decompression is adequately and sufficiently performed, proper subsidence with cages settlement into the vertebral bodies may contribute to the bone fusion process and not adversely affect the clinical outcomes and cervical alignment.Except for subsidence, no other surgical complications were reported, such as those related to the cage, bone-inductive substances, or the anterior plate and screw. Additional surgical treatment for cervical fusion was not necessary throughout the follow-up period. Most previous reports have failed to identify subsidence as a factor predicting low fusion rates, kyphotic change, and unsatisfactory clinical outcomes after surgery.This retrospective study had some limitations. This study included a small number of patients, and there was no control group for comparison of the radiological and clinical outcomes. The enrolled patients in this study were not randomized meaning biased data. Moreover, this study did not identify the factors predicting subsidence and kyphotic changes. Further randomized prospective studies are required to determine the efficacy and toxicity of stand-alone ACDF surgery. In addition, research is needed to identify predicting factors that can cause subsidence and kyphotic changes considering gender and bone mineral density.Stand-alone ACDF does not necessarily provide better outcomes than standard techniques, such as ACDF with plate fixation. Subsidence after stand-alone ACDF occurs to a certain capacity me capacity; however, it does not appear to significantly influence the radiological and clinical outcomes if foramen decompression is adequately and sufficiently provided, as shown in our long-term follow-up study. In contrast, subsidence appears to positively affect the fusion rate in the short-term follow-up.Conceptualization: Moon-Soo Han, Jung-Kil Lee.Data curation: Han-Seung Ryu, Moon-Soo Han, Shin-Seok Lee, Bong Ju Moon, Jung-Kil Lee.Formal analysis: Han-Seung Ryu, Moon-Soo Han, Jung-Kil Lee.Funding acquisition: Jung-Kil Lee.Investigation: Jung-Kil Lee.Project administration: Jung-Kil Lee.Supervision: Moon-Soo Han, Shin-Seok Lee, Bong Ju Moon, Jung-Kil Lee.Writing – original draft: Han-Seung Ryu.Writing – review & editing: Moon-Soo Han, Jung-Kil Lee.


Potatoes are developed vegetatively from tubers, and therefore potato virus transmission is always a possibility. The potato leafroll virus (PLRV) is a highly devastating virus of the genus Polerovirus and family Luteoviridae and is regarded as the second-most destructive virus after Potato virus Y. Multiple species of aphids are responsible for the persistent and non-propagating transmission of PLRV. Due to intrinsic tuber damage (net necrosis), the yield and quality are drastically diminished. PLRV is mostly found in phloem cells and in extremely low amounts. Therefore, we have attempted to detect PLRV in both potato tuber and leaves using a highly sensitive, reliable and cheap method of one-step reverse transcription-recombinase polymerase amplification (RT-RPA). In this study, an isothermal amplification and detection approach was used for efficient results. Out of the three tested primer sets, one efficiently amplified a 153-bp product based on the coat protein gene. In the present study, there was no cross-reactivity with other potato viruses and the optimal amplification reaction time was thirty minutes. The products of RT-RPA were amplified at a temperature between 38 and 42 °C using a simple heating block/water bath. The present developed protocol of one-step RT-RPA was reported to be highly sensitive for both leaves and tuber tissues equally in comparison to the conventional reverse transcription-polymerase chain reaction (RT-PCR) method. By using template RNA extracted employing a cellular disc paper-based extraction procedure, the method was not only simplified but it detected the virus as effectively as purified total RNA. The simplified one-step RT-RPA test was proven to be successful by detecting PLRV in 129 samples of various potato cultivars (each consisting of leaves and tubers). According to our knowledge, this is the first report of a one-step RT-RPA performed using simple RNA extracted from cellular disc paper that is equally sensitive and specific for detecting PLRV in potatoes. In terms of versatility, durability and the freedom of a highly purified RNA template, the one-step RT-RPA assay exceeds the RT-PCR assay, making it an effective alternative for the certification of planting materials, breeding for virus resistance and disease monitoring. Solanum tuberosum L.) is regarded as the most important non-cereal crop in the world. The potato is believed to have its origin 8000 years ago in the high hills of the Andean Mountains of South America and it has become popular worldwide [Myzus persicae), but the virus is also carried by Aphis gossypii. Macrosiphum euphorbiae, which spreads to potato strains less successfully [The potato , we examined three simple RNA isolation procedures to reduce the cost and make RPA easier ,34,41. BThe three primers utilized in the current experiment successfully amplified the fragments of 249 bp (LRRPAF1/R1), 246 bp (LRRPAF2/R2) and 153 bp (LRRPAF3/R3), which was observed using cDNA by two-step RT-PCR under a thermal cycler in the PLRV of infected leaf samples . After iTo make the protocol simple and cost-effective, one-step RT-RPA was standardized. PLRV was successfully amplified at temperatures of 38, 40 and 42 °C when using purified RNA as a template under isothermal conditions (heating block/water bath). However, when disc paper-based RNA was used as the template, out of four temperatures evaluated, no amplification was observed at 36 °C, while PLRV was successfully amplified at 38, 40 and 42 °C. In healthy controls, there was no amplification. It is clear from the results that the PLRV can be identified satisfactorily in one-step RT-RPA at 38–42 °C . BearingTo standardize the protocol experiments further, experiments regarding the minimum time requirement and optimum concentration were performed. An incubation time of 10 min was reported to be sufficient for the detection of PLRV in two-step RT-RPA; however, 20–30 min incubation was required for one-step RT-RPA, which can be seen in Although in a previous report  the cell−0 to 10−8. Subsequently, a comparative analysis with the previous protocols of RT-PCR was performed [−0 to 10−8 for a similar comparative analysis. Agarose gel electrophoresis with concentrations of 2.5% (RT-RPA) and 1.0% (RT-PCR) revealed the limit of detection at various dilutions. We observed almost equivalent sensitivity of both the detection techniques/assay using primer LRRPAF3/R3  was subjected to a serial dilution ranging from 10erformed . AdditioRPAF3/R3  in compaopies/µL .NTN, PVYNT, PVYO, groundnut bud necrosis virus (GBNV) and potato spindle tuber viroid (PSTVd) during the specificity analysis experiment , potato virus M (PVM), PVS, tomato Leafcurl New Delhi virus (ToLCNDV), PVX, PVYperiment .The amplified target amplicons were extracted from the gel, then cloned and sequenced for accurate verification. The amplicon, which was isolated from the field, exhibited 100% sequence similarity with Indian PLRV isolates and other PLRV strains reported from Europe, Canada, Africa, America and the Czech regions. However, this 153 bp region was 98–99% similar to the Czech isolate and Spain isolate. The present results significantly validated the superiority and aptness of the RT-RPA assay. Interestingly, the other viruses were not detected in the specificity analysis experiments.For the study, 129 PLRV-suspected plant parts (asymptomatic and symptomatic) of widely cultivated and popular potato cultivars across different agroecological zones in various states in India were taken to test the optimized one-step RT-RPA method in the field . As a heSimilarly, 10 tubers were tested randomly out of 129 tubers (dormant and stored) and a sharp band (positive) was observed in nine samples using previously reported primers and primer set C using purified RNA as a template in the one-step RT-PCR and RT-RPA, respectively . HoweverA detailed comparative performance analysis of DAS-ELISA, one-step RT-PCR and one-step RT-RPA on leaves, dormant tubers and sprouted tubers (in the case of DAS-ELISA) is shown in During the course of this research, a reliable one-step RT-RPA assay was designed, and it was found to be suitable for the testing of PLRV on a wide scale in seed potato production. Seed tubers are the primary means by which viruses are disseminated. The fact that the established RT-RPA may be used for tubers confers further value of this work.When compared to PCR, RT-PCR and ELISA tests ,36,37, RTo achieve optimal primer specificity in RPA, it is necessary to analyse multiple different sets of primers ,33,34,38The use of a one-step RT-RPA eliminates the necessity of performing separate phases of cDNA synthesis, which in turn lowers the expense of additional chemicals required for cDNA synthesis. Our methodology resulted in a considerable decrease in the amount of reaction volume required (11 µL as opposed to 50 µL) while maintaining the same level of sensitivity. Even though there is a larger upfront cost associated with RT-RPA chemicals, the overall cost of the test has been greatly decreased. Our findings are in agreement with a number of recently published papers ,34,38,39The one-step RT-RPA assay that was developed for this work was put to the test on 129 field samples (one of each leaf and tuber), and when the results were compared to those of the standard DAS-ELISA and RT-PCR, they were found to be extremely encouraging. The number of positive samples found in leaves and tubers utilising the one-step RT-RPA detection approach was reported to be significantly higher than that of RT-PCR. The RT-RPA showed a greater sensitivity that was comparable to RT-PCR while also having a higher level of specificity, as shown in In contrast to the one-step RT-RPA method, the RT-PCR procedure is more vulnerable to the inhibitory chemicals that can be found in the extract of potato leaves and tubers. Additionally, a careful evaluation of various PLRV detection techniques, including DAS-ELISA, one-step RT-PCR  and one-w/v) in PBS-Tween buffer to conduct the DAS-ELISA (pH 7.4). The plates were then kept at 37 °C for two hours after the antiserum had been diluted to a ratio of 1:200 (v/v) in a buffer for the coating that had a pH of 9.6. After incubation, PBS-Tween was used to clean the plates and sap extract was added to each plate well. After that, the plates were heated to 37 °C for two hours, and then they were given a second washing with PBS-Tween. After incubating at 37 °C for two hours, alkaline phosphatase-antiserum was diluted to 1:200 in PBS-Tween. After the final wash, 1 mg/mL of p-nitrophenyl phosphate disodium was added to the substrate buffer. After incubation for one hour at room temperature, an ELISA reader was utilized to check for any responses at a wavelength of 405 nm . The values of optical density considered to be positive were those that were greater than three times the background level on average. The average background levels were determined for each ELISA plate by conducting tests on at least two wells containing all of the reagents except the sap extract. As a healthy control for the experiment, plants that had been grown from virus-free tissue cultures were utilized. Nuclease-free water was used in place of template RNA to conduct the non-template control, also known as the negative control.The infected potato plants were grown in the live virus culture maintenance facility at ICAR-Central Potato Research Institute (ICAR-CPRI) in Shimla, Himachal Pradesh, India. Both the RT-PCR (reverse transcription polymerase chain reaction) and the DAS-ELISA (double-antibody sandwich enzyme-linked immunosorbent test) employing a specific antiserum  demonstrated the presence of PLRV. Leaf or sprouting tissue was carefully crushed and diluted with 1:10  or 2 μL of cellular disc paper-based RNA extract [TM First-strand cDNA synthesis kit, Life Technologies, USA). An amount of 25 mg of leaf tissue was crushed in 1 mL of extraction buffer #2  Triton X-100, 1% (v/v) Tween 20) for cellular disc paper-based RNA extraction. In a 0.2 mL PCR tube, a 3 mm-diameter disc of Whatman Grade 1 filter paper was placed. Then, 100 μL of leaf tissue homogenate was added and the tube was left at room temperature for 5 min before the mixture was removed and discarded. To clean the cellular disc, 200 μL of wash buffer  Tween-20) was utilized. The whole RNA was then eluted at 94 °C for 5 min and kept at −80 °C in 35–50 μL of RNase-free water for further use. Using a Thermo ScientificTM NanoDrop 2000, the quality and quantity of cDNA and RNA were assessed . For cDNA, the absorbance ratio of 260/280 was 1.8–1.9, whereas, for RNA, it was 2.0–2.1.Total RNA was isolated from both PLRV-infected and healthy plant sections using the Spectrum extract  was usedw/v) agarose gel stained with ethidium bromide solution in 1X TAE buffer at 100 volts for 1 h, along with a DNA ladder, and could be seen under UV light.An Applied Biosystems GeneAmp9700 PCR system was used to perform the RT-PCR-based PLRV detection using a method described previously . The 452https://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi (accessed on 30 June 2021)) by following TwistAmp®, Aylesford, UK, reaction kit’s instructions (https://www.twistdx.co.uk (accessed on 30 June 2021). Based on sequences found in the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/blast (accessed on 30 June 2021), the conserved region of the complete coat protein gene was chosen for making primers . http://www.ncbi.nlm.nih.gov/blast (accessed on 30 June 2021) was used to look at the specificity of primers in a workstation. All three primer sets  Limited, India) were tested to see how well they worked with the thermal cycler for RT-RPA  and then 0.5 µL of magnesium acetate was added to each tube. The mixture was heated at 40 °C for 4 min on equipment such as a heating block, water bath or thermal cycler. The mixture was again mixed with a pipette and kept on the same apparatus. After 30 min of incubation, the reaction was stopped by keeping the tubes at 65 °C for 10 min. The visualization on a 2.5% gel gave the desired results. The single best primer was selected for subsequent experimentation only after visualization of the amplified product at different temperature regimes .The three primer sets  were employed in this investigation to search for PLRV, and the cDNA served as a template for RT-RPA. Based on its sensitivity and specificity, the primer set C (LRRPAF3/R3) was chosen for future research and the precise detection of PLRV. First, the freeze-dried reaction pellets were rehydrated with rehydration buffer (29.5 µL) and 8.2 µL nuclease-free water from the TwistDx, TwistAmpTo make the reaction even easier, 0.5 μL of reverse transcriptase (200 U/μL), 0.5 μL of RNase Inhibitor (20 U/μL) and 1 μL of total RNA as a template were added to a one-step RT-RPA. The rest of the components were the same as the ones in the RT-RPA with cDNA, which was described in the section before this one. RNA was used as a template and RT-RPA was performed in one step at different temperatures . Based on the results, the next set of tests was performed at 40 °C. In RT-RPA reactions, the best incubation time  was also analysed. Then, for further experiments, an incubation time of 30 min was chosen for one-step RT-RPA. Standardization was also performed on the amounts of magnesium acetate . All of the tests were performed three or four times. A healthy control was also used to see if the RT-RPA showed any non-specific amplification.TM Plant Total RNA kit . We investigated several concentrations of sodium sulphite  in simple RNA isolation from potato leaves and tubers to increase the quality of the RNA because the method itself was not very good at obtaining RNA from leaves and dormant tubers in the case of PLRV. To compare RT-PCR- and RT-RPA-based PLRV detection, purified RNA from the SpectrumTM Plant Total RNA kit  was also employed. The RNA preparation technique utilizing cellular disc paper was used to detect PLRV in potato leaves in light of the findings.As crude RNA extraction in PLRV was difficult due to its phloem limiting factor and the high concentration of polyphenols and polysaccharides in potato leaves and potato tuber, we tested earlier reported simple RNA isolation procedures to reduce the cost and make RPA easier ,34 to ma−0 to 10−8. Subsequently, a comparative analysis with previous protocols of RT-PCR was performed [−0 to 10−8 for a similar comparative analysis [https://cels.uri.edu/gsc/cndna.html (accessed on 5 June 2022). The initial copy number of the plasmid was 3.276 × 1010. A one-step RT-RPA assay was used to test the specificity of the method utilizing RNA taken from PVA, PVM, PVS, PVX, PVYNTN, PVYNT, PVYO, GBNV and PSTVd infected plants as well as a healthy control plant, and DNA isolated from ToLCNDV infected plants. Gel electrophoresis on agarose gels with concentrations of 2.5 and 1.0% for one-step RT-RPA and RT-PCR, respectively, was used to determine the detection limit.For the sensitivity assay of one-step RT-RPA, the initial template of total RNA (1 μL) was subjected to serial dilution ranging from 10erformed . Additioanalysis ,34. DepeTM Gel Extraction Kit , the visualized amplicons on the agarose gel were extracted and then further cloned in pJET1.2/blunt plasmid vector  in Escherichia coli JM107. Following the confirmation of the positive clones by the colony PCR, sequencing was carried out on an ABI PRISMTM 310 genetic analyser  using M13 forward and reverse primers. Using BLAST, which can be found at http://www.ncbi.nlm.nih.govt/blast (accessed on 25 November 2022), the sequences that were obtained were analysed in the NCBI database.In order to conduct a specificity analysis, an RT-RPA amplification using the designed primers was carried out. Using a GeneJETThe robustness of the optimized one-step RT-RPA method was further validated by testing 99 PLRV suspected field samples (leaves and tubers) of popular potato cultivars during the year 2019–2020. These samples were collected from fields across the states of India. The states of India that are believed to have contributed the suspect samples are as follows: Bihar, Gujarat, Haryana, Himachal Pradesh, Madhya Pradesh, Meghalaya, Punjab, Uttar Pradesh and West Bengal. Virus-free tissue culture-grown mother plants and mini-tubers were used as the samples for determining the overall health of the population. The validation procedure also made use of the tubers that had been saved from diseased plants. In addition to examining dormant tubers (without sprouts), sprouts and leaves, a comparison between DAS-ELISA and one-step RT-PCR/RT-RPA was conducted. Comparable results from this investigation were discovered . The RT-In order to detect PLRV in leaf and tuber samples, a one-step RT-RPA assay that is efficient and simple to use, in addition to being highly sensitive and specific, has been devised. The established technique can be carried out using only a small quantity of rapidly extracted simple RNA from fresh or preserved materials; purified RNA is not necessary for the analysis of leaf samples. In summary, the established method can replace conventional RT-PCR in several situations, including the screening of micro-propagated plants, disease-free planting material certification programmes and the screening of gene pools for PLRV resistance. Regarding the topical goal of resource optimization, this technique of PLRV detection utilizing simple RNA extracts of leaves and tubers followed by a single step of RT-RPA is promising and, to the best of our knowledge, is being documented here for the first time.


COVID-19 has affected older persons the most. The propensity to have severe COVID-19 or die of the infection was especially prevalent among older subjects with multimorbidity, frailty and sarcopenia. The aim of our study was to check which of the simple clinical biomarkers, including the assessment of muscle and frailty, would associate with the survival and the length of hospital stay in older patients with COVID-19. An additional aim was to report the influence of chronic diseases, chronic medication use, and COVID-19 signs and symptoms on the aforementioned outcome measures.The CRACoV study was a prospective single-center  observational study of clinical outcomes in symptomatic COVID-19 patients that required hospital treatment. We analysed data of persons aged ≥ 65 years. We assessed muscular parameters in accordance with EWGSOP2, frailty with the Rockwood Clinical Frailty Scale. We used the data of the initial and 3-month assessment. Demographic characteristics, past medical history, and baseline laboratory values were gathered as a part of routine care. We calculated sex and age, and additionally number-of-diseases adjusted odds ratios of mortality associated with studied factors and betas of the relation with these factors and the length of hospital stay.p = 0.003) greater risk of death, and 16.8 h longer hospital stay (p = 0.01). One score greater Rockwood was associated with 86% (p = 0.002) greater risk of death, but was unrelated to the length of hospital stay. Hand grip strength and dynapenia were unrelated to mortality, but dynapenia was related to longer hospital stay. Probable sarcopenia was associated with 441% (p = 0.01) greater risk of death.The mean  age of 163 participants  was 71.8 (5.6) years, age range 65–89 years. One score greater SARC-F was associated with 34% (In conclusion, the patient assessment with SARC-F and the Rockwood Clinical Frailty Scale may significantly improve the prediction of outcomes in older patients with COVID-19 and by extension might be of use in other acute severe infections. This, however, requires further research to confirm. COVID-19 has affected older persons the most. The propensity to have severe COVID-19 or die of the infection was especially prevalent among older subjects with multimorbidity, frailty and sarcopenia . What thSkeletal muscle undergoes a profound change with ageing. When pathologically advanced, this change may assume a form of dynapenia, namely low muscle strength . When itThere are several tools to assess muscle mass, strength, and performance . Some ofThere is still a relative paucity of data concerning the acute change, and its direction (i.e. increase or decrease), of muscle mass and strength in hospitalized older patients with COVID-19. Likewise, relatively little is known about the relation between simple clinical measures of sarcopenia, dynapenia and muscle strength on one hand and mortality and length of hospital stay due to COVID-19 on the other, especially, in the context of diseases, medications, and symptoms of COVID-19. Still less data are available pertaining to relatively healthy older persons, with good overall functional status. This may be of special importance as deaths from COVID-19, or the development of the post-COVID-19 syndrome, occurred also in young and apparently healthy and fit persons .The aim of our study was to check which of the biomarkers, including simple clinical biomarkers for the assessment of muscle-related health, frailty, chronic diseases, chronic medication use, and COVID-19 signs and symptoms would be associated with risk of poor survival and the increased length of hospital stay in older patients with COVID-19.2). We collected information concerning the signs and symptoms of COVID-19, the data enabling the disease severity assessment according to the National Institutes of Health (NIH) classification , and chronic medications and comorbidities  vs. 1 [0–7] p = 0.007), greater Rockwood Clinical Frailty Scale , and greater number of diseases . The mean BMI was 29.1 (5.1) kg/m2, the calf circumference was 36.8 (4.3) cm, and the hand grip strength was 23.3 (10.8) kg . There were no differences in initial median pO2 (91 [80–100]%). Among survivors, by the 3-month follow-up visit, hand grip strength increased by 5.3 (6.0) kg in women, and 9.2 (8.7) kg in men (p for change and between gender comparison < 0.0001). Dynapenia was present in 40.3% participants, and its frequency did not significantly differ between those who died and survived . At baseline patients on average (SD) had a SARC-F score of 1.9 (2.5), range 0–10. Based on SARC-F 22.2%, and based on SARC-F combined with dynapenia 14.7%, patients had suspected, and probable sarcopenia, respectively. The corresponding values for Rockwood Clinical Frailty Scale was 2.0 (1.3), range 0–6. Based on Rockwood Clinical Frailty Scale 29.6% of patients were pre-frail or frail. The baseline severity of the disease (NIH) was 2.2 (0.9) and it did not differ between survivors and non-survivors. The median (5th-95th percentile) admission C-reactive protein (CRP) in patients who have died was 93.1 (2.0-221.0) mg/l and was higher than in survivors . Admission interleukin 6 (IL-6) was 55.8 (7.6–1781.0) pg/ml in patients who died and was greater than in survivors . The mean (SD) length of hospital stay was 16.1 (8.3) days The frequencies of particular diseases, signs and symptoms, and medications are contained in Table The mean  age of 163 participants (44.8% women) was 71.8 (5.6) years, age range 65–89 years. Twenty-four (14.7%) died. Patients were on average (SD) burdened with 2.9 (2.2) diseases, ranging 0–14. Multimorbidity was present in 68.5% and was significantly more frequent in persons who died compared with survivors , and Rockwood Clinical Frailty Scale (p = 0.002), in separate logistic regression models, each time with adjustment for sex and age, were related to mortality (Table p = 0.03), calf circumference (p = 0.01), and SARC-F (p = 0.01). One score greater SARC-F was associated with 34% (p = 0.003) greater risk of death, and 16.8 h longer hospital stay (p = 0.01). One score greater Rockwood was associated with 86% (p = 0.002) greater risk of death, but was unrelated to the length of hospital stay. After additional adjustment for number of diseases, mortality was related to SARC-F (p < 0.05), and Rockwood Clinical Frailty Scale (p = 0.05). After additional adjustment for the number of diseases present, none of the measures-of-muscle were related to the length of stay (Table p ≤ 0.03) but was not related to the length of hospital stay. Probable sarcopenia was associated with 441% (p = 0.01) greater risk of death. After additional adjustments (stepwise selection optimised models 2s) probable sarcopenia  retained its significance in prediction of fatal events. Of note, tachypnoea , number of diseases  and active cancer  were significantly associated with the risk of death. When CRP and IL-6 were additionally offered in the stepwise procedure, probable sarcopenia lost its significance. In this model CRP was a significant predictor of fatal outcome . Of the included patients 14.8% died. SARC-F . They were also associated with longer length of hospital stay (both p ≤ 0.03) (Table p ≤ 0.05)  (Table p ≤ 0.05)  Fig. . Of signThe severe acute respiratory syndrome due to coronavirus infection has been linked to the loss of muscle mass and strength . The patThe fact that in our analyses the SARC-F or Rockwood Clinical Frailty Scale seem to have performed better than a direct measure of muscle strength may stem from several factors. First, the information in SARC-F reflected a steady state that preceded the infection and might thus have been a better descriptor of sarcopenia at the moment of hospital admission. Second, SARC-F is influenced by more factors than hand grip strength. It comprehensively assesses both upper and lower extremities, and to a certain degree the overall ability for independent mobility , 18. In We found that in a group of older adults hospitalised due to COVID-19 infection, after three months hand grip strength improved in the survivors. This may reflect the degree to which acute COVID-19 infection may have caused acute dynapenia in the first instance. This is in line with the 12-month observation of a cohort of COVID-19 patients from Belgium .Cheval et al., based on data of 3600 persons from the Survey of Health, Ageing and Retirement in Europe (SHARE) cohort, reported that higher hand grip strength assessed between 2004 and 2017 was associated with lower risk of hospitalisation due to COVID-19 in 2020 . It may Several other reports addressed the issue of muscle mass, strength or function in relation to the outcome of COVID-19 –25. We fGreater number of diseases and presence of multimorbidity were related to mortality and longer hospital stay. Of the single diseases, chronic obstructive pulmonary disease, current cancer, and diseases related to risk of atherosclerosis (diabetes mellitus) or being a consequence of it , and chronic kidney disease were related to higher mortality.Casas-Deza et al. reported that the presence of any comorbidities in older hospitalised patients with COVID-19 has been associated with greater mortality . In a stOther published data indicate that three or more diseases and a Charlson index of ≥ 3 are related to greater mortality in older COVID-19 patients , 36. Thi2 < 95% on room air predicted mortality [2 was related to longer hospital stay but not greater risk of mortality. The possible explanation is that our patients with lower SpO2 values had more severe disease that required longer therapy and monitoring.Surprisingly, in our cohort, most of the signs and symptoms recorded on admission were not related to mortality or length of stay. The exception was tachypnea. This stands in contrast with some other studies which indicated that, productive cough or myalgias, and dyspnoea may among other symptoms be associated with worse prognosis , 44. Theortality . In our In our cohort we found that aspirin and antidepressant use was related to greater mortality. Monserrat Villatoro et al. found that among eight studied medications, aspirin was one of those related to worse outcomes . This isWe found that by 3-month visit hand grip strength increased, which we believe to be a protective phenomenon. Previously, Chou et al. found that greater baseline hand grip strength was found to be associated with less cognitive decline after 10-year follow-up . HashidaOur study needs to be considered in the context of its limitations. First, our group consisted of a relatively small number of consecutive older patients from a larger project, of whom 24 persons died. Thus, although our analyses were prespecified, the sample-size calculation was performed for the effect on the change in hand grip strength. Consequently, the numbers may have been too low for other measures and this may have obscured some of the relations such as the influence of inflammatory markers on mortality. Likewise, some statistically significant results may have been driven by chance that arose from multiple testing. The fact that by default we did not include critically ill patients or severely demented patients means that the selection bias could not have been avoided. This needs to be taken into account when interpreting our results. Our cohort was examined between January and June 2021, and thus other published data gathered during different pandemic waves may have pertained to infections with SARS-CoV-2 variants of different virulence.In conclusion, the patient assessment with SARC-F and the Rockwood Clinical Frailty Scale may significantly improve the prediction of outcomes in older patients with COVID-19 and by extension might be of use in other acute severe infections. This, however, requires further research to confirm.


There is growing evidence that the mechanical properties of extracellular matrices (ECMs), including elasticity and stress-relaxation, greatly influence the function and form of the residing cells. However, the effects of elasticity and stress-relaxation are often correlated, making the study of the effect of stress-relaxation on cellular behaviors difficult. Here, we designed a hybrid network hydrogel with a controllable stress-relaxation gradient and a constant elasticity. The hydrogel is crosslinked by covalent bonds and dynamic peptide-metal ion coordination interactions. The stress-relaxation gradient is controlled by spatially controlling the coordination and covalent crosslinker ratios. The different parts of the hydrogel exhibit distinct stress-relaxation amplitudes but the have same stress-relaxation timescale. Based on this hydrogel, we investigate the influence of hydrogel stress-relaxation on cell spreading. Our results show that the spreading of cells is suppressed at an increasing stress-relaxation amplitude with a fixed elasticity and stress-relaxation timescale. Our study provides a universal route to tune the stress-relaxation of hydrogels without changing their components and elasticity, which may be valuable for systematic investigations of the stress-relaxation gradient in cell cultures and organoid constructions.  Cell behaviors, such as spreading , migratiε) in hydrogels, while the timescale of stress-relaxation depends on the intrinsic kinetics (τc) of dynamic crosslinking ,5857,58]6-Zn2+ crosslinkers. Hydrogel resistance to cellular traction forces almost does not relax over time, leading to fast cell spreading  and thiol-connected RGD peptide (cyclo(-Arg-Gly-Asp-D-Phe-Cys)) were purchased from GL Biochem  Ltd. Unless otherwise stated, all other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. . NCTC clone 929 , HeLa, and MCFs were provided by the Cell Bank of the Chinese Academy of Sciences . Acrylate-terminated 4-armed polyethylene glycol  was purchased from JenKem, Inc., Beijing, China. Acrylate-connected histidine-rich peptides  was added to all the solutions as the photoinitiator at a concentration of 0.5 mg mL−1. Hydrogel precursors were injected into band-shaped molds before polymerization under UV illumination  for 2 h at room temperature. Transparent hydrogels were obtained and extensively dialyzed in deionized water to remove the unreacted monomers. Acrylate-PH2. The Young’s modulus of these hydrogels was determined every 15 min for the next 9 h (E) of the hydrogel after being immersed for different times (t) can be summarized as E = 19.62t + 197.73.Then the hydrogels were cut into 20 pieces and immersed in Tris buffer solution  containing 2.2 mM ZnClnext 9 h . The You6-peptides, acrylamide, and 4-armed PEG-Aclt were dissolved in Milli-Q water and mixed to prepare the precursors. Three kinds of hydrogel precursors at different concentrations of 4-armed PEG-Aclt  were prepared, while the concentrations of acrylate-PH6 peptides and acrylamide remained at 60 and 225 mg mL−1. All the solutions were degassed under the protection of argon and sonicated for 3 × 5 min to remove the dissolved oxygen. LAP was added to all the solutions as the photoinitiator at a concentration of 0.5 mg mL−1. Then, the three kinds of hydrogel precursors were injected into band-shaped molds layer by layer before polymerization under UV illumination  for 2 h at room temperature. Transparent hydrogels  were obtained and extensively dialyzed in deionized water to remove the unreacted monomers. Acrylate-PH2 to trigger the formation of gradient coordination. The descent speed was kept at ~0.166 mm min−1 according to the correlation between Young’s modulus and the immersion time. For the hydrogels used for cell culture, acrylate-terminated RGD peptide was added to the precursor to a concentration of 1.0 mg mL−1, and the hydrogels were prepared as described above.Then, the band-like hydrogel was held by a clamp and gradually descended into Tris buffer solution  containing 2.2 mM ZnClScanning electron microscopy (SEM) images were obtained using a Quanta scanning electron microscope  at 20 kV. The hydrogels were lyophilized prior to the measurement. Then, all the samples were sputter-coated with platinum and imaged.−1 to 0.35 mm min−1  depending on the original thickness of the hydrogel. For the compression-relaxation cycle test, the compression rate was also maintained at 0.33 to 0.35 mm min−1  depending on the original thickness of the hydrogel, and the compression strain was maintained at 0.8 to 0.9 mm min−1  depending on the original thickness of the hydrogel. For the stress-relaxation experiments, a compressive strain of 50% was applied to the hydrogels quickly (in ~0.2 s), and then the relaxation of the stress was recorded. The characteristic relaxation times were determined by fitting the stress-relaxation curves with an exponential model.The mechanical properties of the hydrogel were determined using a tensile-compressive tester (Instron-5944 with a 10 N sensor) in the air at room temperature, and the illustration is shown in V1. Then the hydrogels swelled in deionized water for 24 h and immersed in Tris buffer solution  containing 2.2 mM ZnCl2 for different immersion times. Then, the volume of the hydrogels was recorded as V2. The swelling ratios of typical locations on the SRG hydrogel were equal to V2/V1.Five typical locations on band-shaped hydrogel were cut, and their original volumes were recorded as W1. Then the hydrogels were lyophilized, and the weights were recorded as W2. The water content of the hydrogels was equal to (W1 − W2)/W1.The dry weights of hydrogels after swelling were recorded as L929, HeLa, and MCFs cells were cultured in 89% standard Dulbecco’s modified Eagle’s medium . Then, 10% bovine serum  and 1% penicillin/streptomycin  were added to the medium. The medium was changed every 3~4 days.5 mL−1. The incubation of cells was performed at 37 °C and 5% CO2. After 24 h, cell viabilities were evaluated using the Cell Counting Kit-8, following the manufacturer’s instructions .For the cell culture on hydrogel, L929, HeLa, and MCFs cells were trypsinized using 0.05% trypsin (Invitrogen) and washed using serum-free DMEM. Then, the cells were seeded on the hydrogels at a density of 10The L929 cells culture medium was first removed from the hydrogels. Then, L929 cells on the hydrogel were fixed with 4% paraformaldehyde at 37 °C for 10 min. L929 cells were then washed three times in PBS containing calcium (cPBS) and incubated for 30 min in 1% Triton. The samples were stained with 0.1% phalloidin  for 90 min and DAPI for 1 min . Finally, images were obtained using an OLYMPUS-IX73 fluorescence microscope . Cell morphology analysis was performed using ImageJ.t-test. Statistical significance was set to a p-value < 0.05.Statistical significance was determined using the Student’s In conclusion, we developed a hybrid network hydrogel with a constant Young’s modulus and controllable stress-relaxation gradient by altering the coordinate and covalent crosslinker ratios. The gradual stress-relaxation of hydrogels along the coordination crosslinking ratios was studied in detail. Based on the hydrogel, the effects of the amplitude of the stress-relaxation on cell spreading behaviors were investigated. The study in this work indicates that cell spreading is suppressed at an increasing stress-relaxation amplitude with a fixed elasticity and stress-relaxation timescale. We anticipate that the hydrogel can find broad applications in systematic investigations of the stress-relaxation gradient on cell behaviors. The design principle in this study also represents a general route to spatially tune the stress-relaxation of hydrogels without affecting their components and elasticity.


We investigated whether (1) imported pre-cut tissue is feasible for Descemet membrane endothelial keratoplasty (DMEK) in eyes of Asian patients, (2) the clinical outcome is comparable between the endothelium‐in and endothelium‐out methods, and (3) the corneal edema-induced anterior curvature changes may have an effect on the refractive error.The medical records of 32 DMEK patients who underwent either the endothelium-out or endothelium-in method using imported pre-cut grafts with a 3-day pre-cut-to-use time were retrospectively analyzed. Fuchs’ endothelial dystrophy (37.5%) and bullous keratopathy (62.5%) cases were included. The main clinical outcome measures were graft survival, best corrected visual acuity (BCVA), endothelial cell density (ECD), corneal thickness (CT), and complications. Correlation of the anterior curvature changes with refractive error was analyzed in the DMEK with cataract surgery group.2, respectively. The mean survival time, ECD, BCVA, CT, and complication rates were not different between the endothelium-in and endothelium-out methods. A hyperopic shift by +0.42 D was not related to the anterior curvature changes.The overall survival rate was 71.9%. Final graft failures were caused by rejection, glaucoma, and infection. Visual acuities improved by 89.3%. BCVA better than 20/40 and 20/20 was found in 75% and 28.6% of patients, respectively. The ECDs at 3 months and 1 year were 1400 and 1083 cells/mmImported pre-cut tissues with a ≤ 3-day pre-cut-to-use time are feasible for DMEK in the treatment of corneal endothelial edema in eyes of Asian patients, and both endothelium-in and endothelium-out methods appear to be comparatively effective. Edema-induced anterior curvature change may not affect the refractive shift. Since Gerrit Melles first proposed the concept of posterior lamellar keratoplasty in 1998 , 3. AfteSince various surgical techniques are employed in DMEK to reduce the endothelial cell loss , 14–19, Additionally, the refractive outcome and factors causing shift in the refractive errors have been currently debated in patients with FED following DMEK combined with cataract surgery –23. VariOverall, three issues need to be addressed: (1) whether imported pre-cut tissues are feasible for use in DMEK in Asia, (2) whether the clinical efficacy of the endothelium‐in delivery method is comparable to that of the endothelium‐out method in DMEK, and (3) the extent of shift in the refractive error following DMEK and whether it is related to the anterior curvature changes. Therefore, this study investigated 1) the clinical efficacy of DMEK in eyes of Asian patients using imported pre-cut tissues with a ≤ 3-day pre-cut-to-use time, 2) the correlation of the anterior curvature changes with refractive shift in DMEK combined with cataract surgery, and 3) whether the clinical efficacy of the endothelium‐in delivery is comparable to that of the endothelium‐out method in DMEK with imported pre-cut tissues. These results will benefit the standardization of DMEK procedures in eyes of Asian patients using imported pre-cut tissues.th in 2022 were retrospectively reviewed.The study adhered to the tenets of the Declaration of Helsinki. Informed consent was waived by the Institutional Review Board because the study was based on the retrospective review of old charts. Under the approval by the Institutional Review Board of Seoul National University Hospital (IRB No. 2108-026-1241), the medical records of DMEK cases performed by one surgeon (M.K.K) at our tertiary referral center between October in 2016 and May 6Patients who had been diagnosed with corneal endothelial edema due to either FED or BK and had undergone DMEK with ≥6 month-follow up were included. The surgical indication was persistent corneal stromal edema with decreased visual acuity. Among the 35 DMEK cases in the above-mentioned period, three cases were excluded from the analysis. Two were excluded owing to loss in follow-up (< 6 months), and the remaining was excluded because the patient had previously undergone whole corneal transplantation prior to DMEK with a high risk of rejection. Of the total 32 cases, one-third had grade 4 FED with corneal stromal edema , and two-third had BK  , 25. EyeThe following data were collected from the medical charts: demographic information that included the recipient’s age, sex, general medical history, ocular medical and surgical history; and ophthalmic findings that included visual acuity, intraocular pressure (IOP), endothelial cell density (ECD), central corneal thickness (CCT), ocular biometric parameters, and the follow-up period . Concomiin situ on top of the donor corneal stroma in Optisol . The mean pre-cut-to-use period for overseas transport was 59±15 hours. Donor tissue information, including age, graft size, donor ECD, and death-to-use period is shown in A total of 32 transplant grafts were supplied by the Eversight Eye Bank  using the standard procedure , 27. OnlPeripheral laser iridotomy or surgical iridectomy during cataract surgery was performed before DMEK. For patients with a cataract or a pupil anomaly, phacoemulsification with an intraocular lens (IOL) insertion or pupilloplasty was performed concomitantly or sequentially prior to DMEK without any adverse event. Endothelium-out grafts were delivered into the anterior chamber during DMEK using glass Jones tubes  in 17 eyes from October 2016 to March 2019. After August 2019, a Coronet EndoGlide insertion device  became available in Korea; after August 2019, 15 eyes received endothelium-in grafts. The size of the recipient’s Descemet membrane stripping was usually 0.5–0.75 mm larger than that of the donor graft (8.0–9.0 mm).In the endothelium-out technique, the graft was freed from the hinge and spontaneously rolled with the endothelium outward. Using a Jones tube, the graft was aspirated inside the tube with a balanced salt solution  and injected into the anterior chamber through a 3.5-mm corneal or scleral tunnel. The graft was unfolded using tapping procedures and was attached using an air tamponade (80–95%) after confirming that the endothelium was positioned downward by the S mark  28]..28].In the endothelium-in technique, the graft was harvested and tri-folded inward with its endothelium. The graft was pulled into the coronary EndoGlide, which had been filled with the balanced salt solution. The graft was delivered into the anterior chamber in the correct orientation with the endothelium facing downward through a 2.75-mm corneal or scleral tunnel. The graft was unfolded with the assistance of tapping and was attached using an air tamponade (80–95%) with a confirmation of the S mark  14]. Th. Th14]. All the patients received systemic steroid treatment for more than 3 weeks. Topical 1% prednisolone acetate  was administered 4 times a day and the dose was tapered down to 1/day after 12 months. Topical 0.5% moxifloxacin eye drops  were applied 4 times a day.postoperatively measured) [pre-post (CCTpreoperative−CCTpostoperative) or ΔKpre-post (Kpreoperative−Kpostoperative) with MNE was analyzed. In most patients, the preoperative posterior curvature (Kpost) data were not available. Therefore, postoperative Kpost (n = 15) was compared with the Kpost in eyes of normal controls (n = 50) in whom the parameters for cataract surgery were measured using an IOL Master 700 .The following data were collected as the main outcomes and analyzed in each time point in a whole group and were also compared in between the endothelium‐in and endothelium‐out deliveries; graft survival, postoperative best corrected visual acuity (BCVA), ECD, CCT, re-bubbling rate and annual EC loss (ECL) rate. Early and delayed complications were additionally compared between those two groups. Visual acuities measured initially by Snellen charts were converted to logarithm of the minimum angle of resolution (logMAR) values for analysis. The proportions of BCVA ≥20/40, 20/25, or 20/20 were evaluated. Patients with amblyopia, advanced glaucoma, or macular disease that could affect visual acuity were excluded from visual acuity analysis. Increased IOP was defined as IOP > 21 mmHg. ECD was checked by specular microscopy . CCT was measured with anterior segment optical coherence tomography. The postoperative immediate and delayed complications were examined by slit-lamp microscopy . To analyze the changes of corneal astigmatism, axial length (AXL), and mean numerical refractive errors (MNEs) in DMEK combined with cataract surgery (n = 18), an auto-kerato-refractometer , topography , and IOL master  were used. MNEs were calculated by subtracting the value of postoperatively measured refractive error from the value of the predicted refractive error calculated by IOL master .Data are presented as mean ± standard deviation where applicable. Statistical analysis was performed using SPSS Statistics 20.0 . The chi-squared test or Fisher’s exact test was used to compare the two categorical variables, and Student’s t-test was applied to compare the means of continuous variables between two groups. The Kaplan-Meier curves were used to analyze the corneal graft survival after a successful DMEK and a statistical difference in Kaplan-Meier curves was assessed by Log-rank test. Pearson’s bivariate co-relation was used between those ocular biometric parameters. A P value of < 0.05 was considered statistically significant.The baseline characteristics of the donors and recipients are shown in Among the donors’ clinical features, the graft size was significantly larger in the endothelium-in group (8.0±0.4 mm) than in the endothelium-out group . The mean donor death-to-use period in the endothelium-in group (135±8 hours) was similar to that in the endothelium-out group . The mean pre-cut-to-use period did not differ between the two groups . There was no difference in the ECD between the two groups.2 (n = 8). After DMEK, the mean ECD was 1400±480 cells/mm2 at 3 months, 1291±497 cells/mm2 at 6 months, 1086±384 cells/mm2 at 1 year, and 951±252 cells/mm2 at 2 years , and +0.40±1.5 D and +0.45±1.4 D in the endothelium-out and endothelium-in groups, respectively . Postope p>0.05; . MNE als p>0.05; . Mean vaThis study demonstrates that 1) the clinical efficacy and safety of DMEK in eyes of Asian patients using imported pre-cut tissues with a < 3-day pre-cut-to-use time are comparable to those in penetrating keratoplasty for BK, 2) the efficacy of the DMEK endothelium‐in method is comparable to that of the endothelium‐out method, and 3) a hyperopic shift by +0.42 D and a significant reduction of corneal astigmatism were observed. Corneal edema-induced anterior curvature changes may not affect the refractive error. However, the ECL within a month and the early annual rates of ECL were greater than those previously reported , 29–31.Since endothelial keratoplasty, including DMEK, DSAEK, and ultrathin-DSAEK, results in better visual acuity and lower graft rejection than those by penetrating keratoplasty , 3, 7, eInitial ECL at 6 months varies from 21 to 56% in reports, based on the proportion of the included FEDs and the various techniques of graft delivery to the anterior chamber , 36, 37.st year; 16.5±11% vs 17.8±12% at 2nd year, p > 0.05). A high proportion of BK inclusions (62.5%) also contributed to the high ECL at the early stage. However, similar to a DSAEK study using imported corneas [Advanced FED with stromal edema was included in this study, whereas FED eyes without significant corneal edema were included in reports from Western countries , 25, 34. corneas , this st corneas , the surin vitro studies discovered that ECL with the tri-folded endothelium-in method was comparable to that with the scroll-based endothelium-out method [Currently, graft delivery and unfolding procedures need to be standardized to improve the surgical outcomes. Critical steps are still performed in variable ways among different surgeons. To minimize either delivery-associated or tapping-induced ECL, we compared the surgical outcomes between the endothelium-in and endothelium-out deliveries. The visual acuity, ECL, and CCT did not differ between the two methods. Recently, t method –42. Moret method . Since tt method , 43, 44.t method Since the dark pigmented iris blocks visibility to the graft, graft positioning with an appropriate orientation is difficult in eyes of Asian patients. Therefore, the tri-folded endothelium-in method with the assistance of graft-grabbing forceps may be an easier procedure with fewer upside-down placements than the endotheium-out method. In most cases, the scleral tunnel is preferred because of the small dimensions of the anterior chamber. Since the Geuder glass cannula is not currently available in Korea, a modified Jones tube with a 3.5 mm incision or an EndoGlide with a 2.75 mm incision are possible options. Using EndoGlide could be beneficial in maintaining anterior chamber stability during insertion compared to the Jones tube.There were no differences in postoperative complications between the endothelium-in and endothelium-out delivery. One patient developed a pupillary block following re-bubble owing to discharge and early ambulation of the patient. Inferior iridectomy is usually recommended for preventing air- or gas-induced pupillary blocks –47. Howepreoperative and ΔKpre-post indicates anterior curvature changes between before and after DMEK, changes of neither CCT nor anterior curvature correlated with the refractive shift, which supports the study by Augustine et al [post) between the post-DMEK and normal control eyes, suggesting that the graft does not induce a major change. However, we could not evaluate directly whether the individual changes of the posterior curvature could affect the refractive shift. Considering that previous studies show that the posterior cornea curvature correlates with the hyperopic shift of post-DMEK eyes [This study demonstrated a hyperopic shift (+0.42 D) which corresponded with the refractive outcomes (+0.55 ~ +1.12 Diopter) of previous studies following triple DMEK , 52, 53.ne et al . AnotherMEK eyes , 23, 54,post was not measured since the preoperative data were not available. Therefore, further studies to assess the correlation of the Δ Kpost with MNE are warranted. Lastly, DMEK was performed by a professor (MK Kim) who had previous experience of performing approximately 1000 penetrating keratoplasties and 30 deep anterior lamellar keratoplasties. Before starting DMEK, the professor had undergone hands-on training programs by a DMEK wet lab course conducted by the Gerrit Melles of Netherlands Institute for Innovative Ocular Surgery (NIIOS) Academy in Rotterdam. This study reported an incidence of learning curve related-early graft failure of 6.3%, which was comparable to the previous incidences of learning curve related-early graft failure of 7.69 to 4% [This study has several limitations. Owing to the limitations of its retrospective design, some of the data were missing. The small sample size owing to the low popularity of DMEK surgery could have affected the statistical power. However, considering that other studies comparing two groups with similar sample sizes , 42, 55,69 to 4% –62. AlthIn conclusion, performing DMEK using imported pre-cut tissue with a ≤ 3-day pre-cut-to-use period may be a feasible option for either BK or FED in eyes of Asian patients. Additionally, the tri-folded endothelium-in method seems to be comparatively effective to the endothelium-out method for DMEK with imported pre-cut tissue. A hyperopic shift may not be related to the anterior curvature changes.S1 FigData are expressed as mean ± standard error of the mean.(TIF)Click here for additional data file.